Cell surface antigens are often shed from a cell by proteolytic cleavage. The resulting fragments are found circulating in the blood. While circulating shed antigen is often useful for monitoring disease state, it can have a negative impact on the outcome of immunotherapy. For this reason, antibodies that target an extracellular juxtamembrane region of a plasma membrane protein which remains cell-associated following shedding are ideal for immunotherapeutic approaches.
Muc1 (episialin, polymorphic epithelial mucin, PEM, PUM, MAM-6, PAS-O, EMA, NPG, DF-3) and Muc16 (CA-125) are plasma membrane mucins that are upregulated in a variety of malignancies (Jacobs and Bast, 1989; Taylor-Papadimitriou et al., 1999). Both Muc1 and Muc16 are type I membrane proteins comprising: (a) a short cytoplasmic domain (69 amino acids for Muc1, 31 amino acids for Muc16), which interacts with the intracellular signal transduction machinery (Li et al., 1998; Li and Kufe, 2001; Li et al., 2001; Li et al., 2001; Fendrick et al., 1997; Konishi et al., 1994)); (b) a transmembrane domain; and (c) a large, heavily glycosylated extracellular domain. The extracellular domain of both proteins comprises a large region of tandem repeats, with 20 amino acid long tandem repeats for Muc1 and 156 amino acid long tandem repeats for Muc16. Muc1 has a variable number of tandem repeats (from 25 to 100, depending upon the allele) (Devine and McKenzie, 1992; O'Brien et al., 2001; O'Brien et al., 1998; Taylor-Papadimitriou et al., 1999). To date, there is no evidence supporting genetic polymorphism of Muc16. The resulting peptide cores of Muc1 and Muc16 have molecular weights of approximately 125–200 kDa and 2.5 MDa, respectively (O'Brien, 2002).
Muc1 is expressed on the surface of epithelial cells as a heterodimer derived from a common precursor (Ligtenberg et al., 1992; Parry et al., 2001). Proteolytic processing may occur cotranslationally in the endoplasmic reticulum by a kallikrein-like protease (Parry et al., 2001). The extracellular subunit remains non-covalently associated with the subunit containing the transmembrane region and cytoplasmic tail throughout intracellular processing and transport to the cell surface. It is not yet known whether Muc16 is proteolytically processed in a similar manner. However, Muc16 has a conserved furin cleavage site (RXK/RR) in the extracellular domain approximately 100 amino acids away from the transmembrane domain (Bassi et al., 2000; Molloy et al., 1999; O'Brien et al., 2001). Furins are implicated in trans-golgi network proteolytic processing of a number of proteins including cell-surface receptors (Molloy et al., 1999).
Both Muc1 and Muc16 may be used as serum markers for diagnosis and for monitoring the progress of treatment of malignancies. Thus, breast tumors may be diagnosed and the progress of treatment monitored using Muc1 antibody assays (Bon et al., 1997), while anti-Muc16 antibodies such as OC125 and M-11 may be used in cases of ovarian cancer (Cannistra, 1993). The mechanism of shedding (i.e. the release of these mucins or their fragments into the blood or other extracellular fluid) is not fully known. In the case of Muc16 shedding may be regulated by serine/threonine phosphorylation of the cytoplasmic domain of Muc16 in response to EGF stimulation (O'Brien et al., 1998). Although Muc1 is also phosphorylated in response to EGF stimulation, there is currently no evidence for a role of such phosphorylation in the mechanism of shedding of portions of Muc1. It is also unclear whether the shed portion of Muc1 corresponds to the extracellular subunit that is produced by the cleavage of the Muc1 protein in the endoplasmic reticulum, or whether there is an additional cleavage site that is targeted by a stromal protease. Less information is currently available regarding the processing and shedding of Muc16. Sequence information indicates that Muc16, in addition to the potential furin cleavage site, has a potential stromolysin cleavage site (SPLA) located about 50 amino acids upstream from the transmembrane domain, cleavage of which could release the fragment of CA125 that is bound by monoclonal antibodies OC125 and M-11.
Tumor-cell specific monoclonal antibodies conjugated to highly toxic maytansinoid drugs and prodrugs have been shown to be effective in the treatment of tumors in mouse models (Liu et al., 1996). The Muc1 and Muc16 proteins represent attractive sources of epitopes for the development of such antibody-containing conjugates, such as may be termed tumor-activated prodrugs (TAPs), because expression of these epitopes is frequently elevated in tumors (see above).
However, a portion of the total Muc1 or Muc16 expressed by tumor cells is shed into the blood stream as evidenced by the ability to use Muc1 and Muc16 antibodies for monitoring disease state (see above). Clinical trials with naked and drug-conjugated monoclonal antibodies to various target antigens suggest that high concentrations of circulating antigen present in some patients is problematic. (Baselga et al., 1996; Pegram et al., 1998; Tolcher et al., 2001). A high concentration of circulating antigen greatly increases the antibody clearance-rate, resulting in low delivery of the antibody to the tumor. Furthermore, in the case of drug-conjugated antibodies recognizing shed antigen, the increased rate of clearance may result in dose-limiting toxicity in the liver. Although some patients may exhibit relatively low levels of shed antigen, the tandem repeat nature of mucins, such as Muc1 and Muc16, resulting in potentially many epitopes per molecule, make the absolute quantification of shed epitope difficult to accomplish. Thus, with currently available Muc1 and Muc16 antibodies to shed portions of these molecules, patients cannot be reliably evaluated for whether their shed antigen level is prohibitively high for antibody therapy. Therefore, there is a need for antibodies that are specific for epitopes contained in the non-shed portions of Muc1 or Muc16, so that cytotoxic drug conjugates of such antibodies may be efficiently directed to tumor cells even in the presence of high concentrations of circulating shed fragments of Muc1 and Muc16. To date, no antibodies defined as reacting with non-shed, extracellular domains of shed proteins have been reported.
The present inventors have developed antibodies, antibody fragments and conjugates of such antibodies or fragments, methods for preparing and screening such antibodies, diagnostic screening methods and treatment methods using such antibodies and conjugates, which address the above-mentioned shortcomings and problems identified in the prior art. The many advantages of the present invention will become apparent to those of ordinary skill in the art upon reading the following disclosure.